Characterisation of in vivo ovarian cancer models by quantitative 1H magnetic resonance spectroscopy and diffusion-weighted imaging

Magnetic resonance imaging (MRI) and spectroscopy (MRS) offer powerful approaches for detecting physiological and metabolic alterations in malignancies and help investigate underlying molecular mechanisms. Research on epithelial ovarian carcinoma (EOC), the gynaecological malignancy with the highest death rate characterised by frequent relapse and onset of drug resistance, could benefit from application of these molecular imaging approaches. In this study, MRI/MRS were used to characterise solid tumour models obtained by subcutaneous (s.c.) or intraperitoneal (i.p.) implantation of human SKOV3.ip cells in severe combined immunodeficiency (SCID) mice. In vivo MRI/MRS, ex vivo magic-angle-spinning (MAS), and in vitro (1)H-NMR measurements were carried out at 4.7 T, 9.4 T, and 9.4/16.5 T, respectively. MRI evaluation was performed by T1-, T2-, and diffusion-weighted (DW) multislice spin-echo imaging. The in vivo (1)H spectra of all tumour models showed a prominent resonance of total choline-containing metabolites (tCho). Quantitative in vivo MRS of both i.p. and s.c. SKOV3.ip xenografts showed that the mean tCho content was in the 2.9-4.5 mM range, with a mean PCho/tCho ratio of 0.99 ± 0.01 [23 examinations, 14-34 days post injection (dpi)], in good agreement with ex vivo and in vitro analyses. Myo-inositol ranged between 11.7 and 17.0 mM, with a trend towards higher values in i.p. xenografts at 14-16 dpi. The average apparent diffusion coefficient (ADC) values of SKOV3.ip xenografts [1.64 ± 0.11 (n = 9, i.p.) and 1.58 ± 0.03 x10(-3) mm(2)/s (n = 7, s.c.)] were in agreement with values reported for tumours from patients with EOC, while the mean vascular signal fraction (VSF) was lower (≤ 4%), probably due to the more rapid growth of preclinical models. Both s.c. and i.p. xenografts are valuable preclinical models for monitoring biochemical and physiopathological changes associated with in vivo EOC tumour growth and response to therapy, which may serve as the basis for further clinical development of noninvasive MR approaches.     

Localized in vivo 1H NMR spectroscopy of murine tumours: effect of blood flow reduction.

Single voxel 1H localized spectroscopy (PRESS at 300 MHz) was used to monitor physiological and biochemical changes induced by hydralazine (5 mg/kg, i.p.) in murine C3H mammary tumours. In addition to a significant increase (by 52%, maximal at 30 min) in the intensity of the 1.32 ppm signal (predominantly from lactate, consistent with a selective reduction in tumour blood supply by hydralazine), downfield shifts in the resonance frequencies of 1H signals were observed. In particular, the signal initially at 3.24 ppm (total choline, tCho) shifted by 0.050 ppm (maximal at 13 min), whereas water shifted by 0.086 ppm. Lactate intensity and water and tCho resonance frequencies returned to control values at approximately 100 min after treatment. No significant changes in the resonance frequencies of water or tCho were observed over this time period in the tumours of mice given saline. In vitro studies showed that, while the resonance frequency of water was temperature dependent, the main components of the tCho signal (choline, phosphorylcholine and glycerophosphorylcholine) were more than 30-fold less sensitive to temperature. It was concluded that the shift in the water resonance frequency was due to the combined effects of tumour temperature reduction and a paramagnetic shift from increased deoxyhaemoglobin levels, whereas the tCho signal was only affected by the paramagnetic shifts.     

Hypoxia-inducible factor 1 alpha is essential for hypoxic p27 induction in endometrioid endometrial carcinoma

Hypoxia-inducible factor 1alpha (HIF-1alpha) plays an essential role in the adaptive response of cells to hypoxia. The cyclin-dependent kinase inhibitor p27(Kip1) is highly expressed in the normal endometrium but is lost during endometrial carcinogenesis. However, in high-grade cancers, p27 re-expression is observed. We analysed the role of HIF-1alpha in hypoxia-induced expression of p27 in vitro and in vivo in endometrial cancer. Paraffin-embedded specimens from endometrioid endometrial carcinoma (n = 39) were stained immunohistochemically for HIF-1alpha, p27, and Ki67. HEC1B, an endometrial carcinoma cell line, was cultured under normoxic or hypoxic conditions in the presence or absence of transiently expressed short hairpin RNAs targeting HIF-1alpha. Protein expression of p27 and HIF-1alpha was assessed by western blotting. Immunohistochemical staining revealed perinecrotic HIF-1alpha expression in 67% of the cases and p27 staining centrally in the tumour islands, mostly around necrosis, in 46% of the cases. In 50% of the tumours with perinecrotic HIF-1alpha expression, p27 and HIF-1alpha perinecrotic/central co-localization was observed. In these tumour sections, hypoxia-associated p27 expression showed less proliferation around necrosis. Analysis of cultured endometrial carcinoma cells demonstrated that p27 protein expression is induced by hypoxia. This induction was abrogated by transient knockdown of HIF-1alpha using RNAi. Furthermore, hypoxia induced cell cycle arrest in HEC1B cells. We conclude that, in endometrioid endometrial carcinoma, p27 re-expression by hypoxia is HIF-1alpha-dependent and leads to cell cycle arrest. This may contribute to the survival of cancer cells in hypoxic parts of the tumour.     

In vivo 1H MRS in the assessment of the therapeutic response of breast cancer patients

MRI and in vivo MRS have rapidly evolved as sensitive tools for diagnosis and therapeutic monitoring in cancer research. In vivo MRS provides information on tumor metabolism, which is clinically valuable in the diagnosis and assessment of tumor response to therapy for the management of women with breast diseases. Several centers complement breast MRI studies with (1)H MRS to improve the specificity of diagnosis. Malignant breast tissues show elevated water-to-fat ratio and choline-containing compounds (total choline, tCho), and any effect of therapy on tissue viability or metabolism will be manifested as changes in these levels. Sequential (1)H MRS studies have shown significantly reduced tCho levels during the course of therapy in patients who were responders. However, there are challenges in using in vivo MRS because of the relatively low sensitivity in detecting the tCho resonance with decreased lesion size or significant reduction in the tumor volume during therapy. MRS is also technically challenging because of the low signal-to-noise ratio and heterogeneous distribution of fat and glandular tissues in the breast. MRS is best utilized for the diagnosis of focal masses, most commonly seen in patients with ductal-type neoplasms; however, it has limitations in detecting nonfocal masses, such as the linear pattern of tumors seen in invasive lobular carcinoma. Further work is required to assess the clinical utility of quantitative MRS, with the goal of automation, which will reduce the subjectivity currently inherent in both qualitative and semi-quantitative MRS.     

The effect of HIF-1alpha siRNA on growth and chemosensitivity of MIA-paca cell line

PURPOSE: Hypoxia-inducible factor-1alpha (HIF-1alpha) primarily mediates the hypoxic response. HIF-1alpha induction by various stimuli contributes to cell proliferation and survival. To investigate the effect of HIF-1alpha, we used small interfering RNA (siRNA), and expected that cell apoptosis and sensitivity to chemotherapeutic drug increase, when we blocked the HIF-1alpha gene. Thus we performed in vitro and in vivo experiment to clarify the effect of hypoxia-inducible factor-1alpha on tumor growth. MATERIALS AND METHODS: We made control and HIF-1alpha siRNA using vector plasmid and then transfected Mia-paca cell lines with these RNAs. After selection with geneticin, two new cell lines were made, confirmed via immunoblotting. After treating with gemcitabine, each cell line was assayed to confirm the effect of HIF-1alpha siRNA using the cell proliferation assay and capase-3 assay. And then in vivo study was performed using female athymic nude mice. After subcutaneously injecting each new cell lines, intraperitoneal gemicitabine chemotherapy was performed for 3 weeks. During that period, we analyzed the difference of tumor growth rate. RESULTS: The tumor growth of HIF-1alpha siRNA-transfected group was slower than that of the control group both in vitro and in vivo experiment. CONCLUSION: The suppression of HIF-1alpha results in decrease of cell proliferation and increase of chemosensitivity of pancreatic cancer cell line. Therefore, targeting the HIF-1alpha may be useful treatment modality for some pancreatic cancers.     

IL-1alpha,IL-1beta and TNF-alpha secretion during in vivo/ex vivo cellular interactions with titanium and copper

Titanium (Ti) and copper (Cu) were used to evaluate cytokine secretion around materials with different chemical properties. Ti disks were coated with Cu or left uncoated. The disks were inserted subcutaneously in rats for 1, 3, 12, 18, 24 and 48 h. Interleukin-1alpha (IL-1alpha), IL-1beta and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured in vivo around the materials, in sham operated sites, and after ex vivo incubation of surface adherent cells. Ti and Cu revealed distinct cytokine expression patterns. Cu recruited cells showed higher and prolonged release of IL-1alpha than Ti at longer times (>24 h), whereas Ti exhibited a transient IL-1alpha response at earlier periods (<24 h). An early enhanced secretion of TNF-alpha characterized Ti. Low amounts of IL-1beta were found around both materials. Sham site recruited cells produced lower levels of cytokines. The results after ex vivo incubations were similar to those in vivo. This study shows that material chemical properties influence early cytokine production. The Ti-associated transient rise of IL-1alpha and TNF-alpha may be of importance for the early tissue response around biocompatible materials, while a delayed high IL-1alpha expression could be a marker of inflammation induced by toxic materials.     

Radiation-induced changes in gene-expression profiles for the SCC VII tumor cells grown in vitro and in vivo

SCCVII tumor cells that grow in vitro or in vivo as a solid tumor were used to compare and contrast geneexpression profiles with or without exposure to two doses of ionizing radiation. Exponentially growing SCCVII cell cultures and tumors (1 cm diameter) were treated with 0, 2, or 10 Gy, and RNA was collected 1, 3, 6, 12, and 24 h after treatment. Growth under in vitro conditions increased the expression of genes associated with the unfolded protein response (UPR) including ATF4, Ero-1 like, and cystathionase. Growth in vivo indicated that the HIF-1a genes were not upregulated, whereas genes such as hemoglobin alpha and crystallin alpha B were significantly upregulated. Ninety genes of 16K were found to be significantly modulated under either growth condition by radiation treatment. Gene expression was not dose dependent. Sixty percent of these genes exhibited similar modulation under both in vitro and in vivo conditions; however, 29% of the genes were modulated by radiation under in vivo conditions only. Gene-expression profiles for the same tumor cells can differ, dependent on growth conditions, underscoring the influence that the tumor microenvironment exerts on gene expression for both growth of solid tumors and their response to radiation treatment.     

转染缺氧诱导因子-1α对肺癌细胞A549生长的影响

目的:研究缺氧诱导因子-1α(HIF-1α)对人肺癌细胞株A549体内、外生长的影响,并探讨其可能机制。方法:将HIF-1α转染人肺癌细胞A549中,观察A549细胞生长。建立人肺癌裸鼠模型,随机将其分为两组:对照组(A组,10只),HIF-1α转染细胞组(B组,10只)。1月后,切除瘤灶、称瘤重、计算促瘤率。标本用免疫组化测定增殖细胞核抗原(PCNA),同时用Westernblot检测HIF-1α、凋亡抑制蛋白survivin和bcl-2的表达。结果:HIF-1α转染A549细胞后,细胞生长速率加快。转染HIF-1α的裸鼠肿瘤生长较对照组快,A组、B组的瘤重分别为(2.51±0.33)g,(3.44±0.40)g,促瘤率为37%。免疫组化和Westernblot提示:B组的PCNA表达比A组高,B组HIF-1α、survivin、bcl-2的蛋白水平明显高于A组。结论:转染HIF-1α体内、外均可促进肺癌A549细胞的生长,其机制可能与其能促进细胞恶性增殖和抑制凋亡有关。     

In vivo 1H MRS of WSU‐DLCL2 human non‐Hodgkin's lymphoma xenografts: response to rituximab and rituximab plus CHOP

In order to identify early ¹H MRS metabolic markers of response to rituximab immunotherapy and to rituximab plus CHOP (cyclophosphamide, hydroxydoxorubicin, vincristine, and prednisone) combination therapy, we performed an in vivo MRS investigation of a non‐Hodgkin's lymphoma (NHL) xenograft model. Human WSU‐DLCL2 NHL cells were subcutaneously implanted into flanks of female severe combined immunodeficient mice. When tumor volumes reached ～600 mm³, rituximab was administered for three weekly cycles at a dose of 25 mg/kg per cycle with or without CHOP. Before and after treatment, tumor lactate (Lac) and total choline (tCho) were detected using the selective multiple quantum coherence sequence and the stimulated echo acquisition mode sequence, respectively. Rituximab produced a small tumor growth delay (～5 days), whereas treatment with rituximab plus CHOP (RCHOP) led to ～20% tumor regression after three cycles of therapy. After one cycle of rituximab, the tCho/H₂O ratio had decreased significantly (5%, P = 0.003), whereas the Lac/H₂O ratio had not changed (P = 0.58). Both Lac/H₂O and tCho/H₂O had decreased after one cycle of RCHOP treatment (26%, P = 0.001; 10%, P = 0.016, respectively). After two cycles of RCHOP, Ki67 assay of histological tumor specimens indicated ～40% decrease in proliferation (P < 0.001) in the RCHOP‐treated tumors; no change was detected after treatment with rituximab alone. This study suggests that decreases in tCho/H₂O are more sensitive indices of response to rituximab, whereas decreases in Lac/H₂O are more sensitive to response to CHOP combination therapy. Copyright © 2008 John Wiley & Sons, Ltd.     

Cortical spreading depression induces the expression of iNOS, HIF-1alpha, and LDH-A

The mechanisms of tolerance to subsequent episodes of ischemia induced by cortical spreading depression (CSD) are not clear. The effects of CSD on the expression of inducible nitric oxide synthase (iNOS), hypoxia inducible factor-1alpha (HIF-1alpha), and lactate dehydrogenase-A (LDH-A) were evaluated in the present experiment. Unilateral CSD was induced in Sprague-Dawley rats by application of KCl on the right cortex and the mRNA levels of iNOS, HIF-1alpha, and LDH-A were evaluated at 15 min, 2 h, 4 h, 6 h or 24 h after CSD. RT-PCR analysis showed: 1) an increase of iNOS mRNA at 15 min, 2 h, 4 h; 2) an increase of HIF-1alpha mRNA at 6 h; 3) an increase of LDH-A mRNA at 4 h. In situ hybridization with specific digoxigenin-labeled oligonucleotides revealed that the mRNA levels were increased at 15 min-2 h for iNOS, 2-4 h for LDH-A and 6 h for HIF-1 after CSD. Immunohistochemistry analysis revealed that levels of iNOS and HIF-1alpha were increased, respectively, at 2 h and 6 h after CSD. These data suggest that CSD promotes the expression of iNOS, HIF-1alpha, and LDH-A in nervous cells giving a neuroprotective effect.     

Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1)

The hypoxia-inducible factor 1 (HIF-1) plays a critical role in cellular responses to hypoxia. The aim of the present study was to evaluate which genes are induced by hypoxia, and whether this induction is mediated by HIF-1, by expression microarray analysis of wt and HIF-1alpha null mouse fibroblasts. Forty-five genes were up-regulated by hypoxia and 40 (89%) of these were regulated by HIF-1. Of the 114 genes down-regulated by hypoxia, 19 (17%) were HIF-1-dependent. All glycolytic enzymes were strongly up-regulated by hypoxia in a HIF-1-dependent manner. Genes already known to be related to hypoxia, such as glucose transporter 1, BNIP3, and hypoxia-induced gene 1, were induced. In addition, multiple new HIF-1-regulated genes were identified, including genes involved in metabolism (adenylate kinase 4, galactokinase), apoptosis (galectin-3 and gelsolin), and invasion (RhoA). Genes down-regulated by hypoxia were involved in cytoskeleton maintenance (Rho kinase), mRNA processing (heterogeneous nuclear ribonucleoprotein H1 and splicing factor), and DNA repair (REV3). Furthermore, seven cDNAs from genes with unknown function or expressed sequence tags (ESTs) were up-regulated and 27 such cDNAs were down-regulated. In conclusion, hypoxia causes down- rather than up-regulation of gene expression and HIF-1 seems to play a major role in the regulation of hypoxia-induced genes.     

High-resolution magic-angle-spinning 1H NMR spectroscopy reveals different responses in choline-containing metabolites upon gene therapy-induced programmed cell death in rat brain glioma

Changes in the concentrations of choline-containing metabolites (CCM) have been implicated in both cell proliferation and death processes. In this study, high-resolution magic-angle-spinning (HRMAS) 1H NMR spectroscopy was used to study metabolite changes in the CCM chemical shift region in rat glioma ex vivo during apoptosis induced by thymidine kinase-ganciclovir gene therapy. Cell density and apoptotic activity in the tumours were quantified by histological methods. HRMAS 1H NMR was able to resolve peaks from choline (Cho), glycerophosphocholine (GPC), phosphocholine (PC), taurine (Tau) and myo-inositol (myo-Ins), all of which contribute to the in vivo 1H NMR peak centred at 3.23 ppm. The early phase of apoptosis (treatment day 4), with a approximately 2.8-fold increase in the number of apoptotic nuclei (at constant cell density of 1.8 +/- 0.1 x 10(5) cells/mm3) was associated with increases in resonance intensity from GPC and PC, while Cho and Tau remained unchanged. Later stage apoptosis, accompanied by synchronous cell death (cell density declined to 0.7 +/- 0.02 x 10(5) cells/mm3), resulted in a significant decline in Tau relative to untreated tumours, while the contents of CCMs and myo-Ins detectable by 1H HRMAS were unchanged. These observations demonstrate that, while the in vivo 1H NMR peak at 3.23 ppm is indicative of cellular processes involved in apoptosis, the biochemical changes monitored by this resonance involve a number of different and chemically distinct metabolites.